Currently, in various fields including research institutes, medical facilities, inspection agencies, and others, a large number of analytical and detection methods based on a specific base sequence of a target gene are employed. For example, in the case of analyzing and detecting the presence of: a pathogenic microorganism or a minute creature such as a pathogen or allergens including fungus, ticks, and the like; virus; pollen; or the like included in a sample (body fluid or cell fragment) derived from a biological body, such as human and animals, or a sample derived from the environment, methods for detecting a specific base sequence of a target gene included in such detection targets have been used. These methods for detecting nucleic acids (DNA, RNA) can be performed in a shorter period of time and at a higher sensitivity than methods for detecting a protein. Furthermore, even if the amount of nucleic acid originally included in a sample is below a detection limit, the nucleic acid can be amplified relatively easily and specifically using a cell-free system. With these advantages, by using a method for amplifying a nucleic acid or in combination with a method for amplifying a nucleic acid, many methods for detecting a specific base sequence of a target gene have been developed.
Currently, as a nucleic acid amplification technique which are most commonly used, a PCR method is cited in which a cycle of template denaturation, primer annealing to the template, and a DNA polymerase elongation reaction is performed several tens of times by use of a temperature cycle so as to amplify a nucleic acid in a region sandwiched by a primer pair. However, in a method for amplifying a nucleic acid by use of the temperature cycle, an instrument for controlling the temperature cycle (thermal cycler or the like) is expensive and also it is required to examine and set an optimal temperature cycle for nucleic acid amplification.
Therefore, in recent years, a nucleic acid amplification method has been studied in which a DNA polymerase elongation reaction is carried out under a constant temperature condition without using a temperature cycle. A representative example of such a method is a LAMP (Loop-Mediated Isothermal Amplification) method (Patent Document 1). In the LAMP method, four primers are designed for six regions on a target gene so that a 3′ end of a DNA strand to be synthesized always forms a loop and serves as an origin of replication of the next DNA amplification reaction, and a nucleic acid molecule of the target gene is amplified.
Furthermore, in Patent Document 2, described is a method in which a linear DNA molecule having, at the 5′ end and 3′ end, base sequences respectively complementary to two non-continuous regions adjacent to each other in a target gene and having a linker region not complementary to the target gene is designed as a probe, the probe is hybridized to the target gene to form a loop, the juxtaposed 5′ end and 3′ end are connected by a ligase so as to be circularized, and the base sequence of the circularized probe is amplified by using a primer complementary to the linker region.
However, these existing methods still have problems in terms of cost, design and operation of a detection method, and detection sensitivity; therefore, a new nucleic acid amplification method and a detection method which fully meet these problems is needed.
Patent Document 1: International Patent Publication No. WO 2000/28082
Patent Document 2: Japanese Patent Translation Publication 2005-508599